Project Summary/Abstract Remodeling of neural circuits is essential for development, learning, and normal brain function, and intimately dependent on astrocytes and microglia -- the glial support cells of the brain. The goal of this proposal is to identify novel glial-encoded genes that promote neural circuit remodeling and to design glial-based tools to track and manipulate brain plasticity in vivo. The central premise of this proposal is that microglia, the resident phagocytes of the brain, respond to cues from local astrocytes, stromal cells that are intimately associated with neuronal synapses. Together these cells coordinate a tissue remodeling response, physically clearing space to enable new synapse formation. We predict that this coordinate regulation between the stromal and immune cells of the brain will co-opt innate immune pathways that are increasingly implicated in tissue remodeling elsewhere in the body. This model will be tested in the mouse barrel cortex in which whisker removal early in postnatal life leads to plasticity and topographic rearrangement of neuronal inputs. This is an inherently heterogeneous response that requires local detection of altered sensory inputs, removal of superfluous synapses, and formation of new synapses. Our experimental design will proceed in three phases. First, we will perform single cell RNA sequencing of astrocytes and microglia during barrel cortex structural remodeling to identify coordinate regulation modules: ligand-receptor pairs that are upregulated or alternately spliced in astrocytes and microglia respectively during barrel cortex remodeling. Our top candidates will be misexpressed or deleted during barrel cortex plasticity via adeno-associated virus (AAV) delivery of transgenes or CRISPR/Cas9 genome editing constructs. We predict that the top candidate pathways will be heterogeneously expressed in remodeling barrel cortex in situ and will impact cortical plasticity when misexpressed in vivo. Our ultimate goal is to design new glial-based tools to both report and regulate neural circuit plasticity that can be used to study less experimentally accessible circuits throughout the nervous system, particularly those involved in cognition and behavior. We anticipate that prospective identification of glial-encoded plasticity pathways will have broad ranging applications for understanding and modulating neural circuits in the context of development, injury, and neuropsychiatric diseases.